1. Field of the Invention
The present invention relates to an optical coherence microscope (OCM) for study of problems in developmental biology and biotechnology. More particularly, the invention is used for imaging cells located up to four millimeters or more below the surface of living tissue.
2. Description of the Related Art
Optical coherence microscopy (OCM) is a technique developed recently to image objects embedded in an opaque medium (e.g., flesh) up to a depth of 1 to 2 mm. It has been applied successfully on a prototype basis in ophthalmology (Swanson et al., 1993) and dermatology (Schmitt et al., 1995) to image tissue structures and interfaces. Moreover, OCM has been used to measure the optical properties of tissue and thereby provide information on the physiological state of tissue. OCM has recently become a subject of interest for the study of developmental biology.
Understanding of developmental mechanisms has come from studies of gene expression patterns, tissue geometry, and/or cell morphology, all performed on fixed tissue. From these xe2x80x9csnap-shotxe2x80x9d views, researchers must infer the dynamics of the underlying cellular and molecular events. Recently, biological imaging technologies have been introduced that permit the non-destructive analysis of cell migration, differentiation, and neuronal interconnection during embryonic development. For example, fluorescent or absorbing compounds can be used to label cells which are then followed with a conventional light microscope equipped with a video camera or with a confocal microscope. The confocal microscope adds significant depth resolution, offering the possibility of obtaining a three-dimensional image by combining optical sections through the depth of an embryo. The image formation rate of the confocal microscope is sufficiently fast to follow the dynamic behaviors of cells as they migrate or of retinal cell axons as they extend, actively sense, and retract projections toward cells in the tectum (O""Rourke et al., 1994). However, light scattering in embryonic tissue reduces the signal-to-noise ratio of a confocal microscope, limiting the depth of the specimen that can be explored to about 200 xcexcm (Schmitt et al., 1994b). A second imaging technology is magnetic resonance imaging (MRI), recently extended to the microscopic domain so that it can now resolve a 12 xcexcm cube in living embryos (Jacobs and Fraser, 1994). Although an MRI microscope is indifferent to optical opacity, it is both expensive and slow, requiring nearly an hour to generate a high-resolution image.
It is worth noting that other recently developed imaging techniques also experience image degradation with depth into tissue. For example, green fluorescence protein (GFP) has been modified and expressed in the plant Arabidopsis thaliana, yielding beautiful images of developing roots. However, the images are obtained with a confocal microscope and are limited to depths less than 100 xcexcm in this preparation. Development of the primary meristem in the seed embryo occurs several hundred micrometers into the tissue, too deep for confocal microscopy. Similar limitations apply to 2-photon microscopy (Potter et al., 1996) and fluorescence resonance energy transfer (Helm and Tsien, 1996).
Optical Coherence Microscopy
An optical coherence microscope uses the principles of confocal microscopy, with an additional coherence gate that excludes back-scattered light from out-of-focus planes, resulting in a signal-to-noise ratio that is enhanced by 6 orders of magnitude (lzatt et al., 1994a,b). A resolution of 10 xcexcm has been achieved in both the lateral and depth directions (Huang et al., 1991b). Optical fiber and solid state sources/detectors are typically used, so the instrument is inherently rugged. OCM overcomes the depth limitation of confocal microscopy and is currently faster than MRI. And at an estimated cost of under $10,000 the instrument is two orders of magnitude less expensive than the MRI microscope.
The coherence gate in OCM is achieved by superposing a Michelson interferometer on the confocal microscope. Back-scattered light from the specimen interferes coherently with light returning from an added reference arm only when the two optical paths are equal. The amplitude of interference fringes (their xe2x80x9cvisibilityxe2x80x9d) becomes the signal; this signal is appreciable only for light back-scattered from a narrow range of depths in the specimen. The depth range over which interference occurs is related to the coherence length of the source. For example, the depth range, which is also the depth resolution, is roughly 10 xcexcm when the spectral width of the source is 30 nm (xcex=830 nm, Swanson et al., 1992). At a particular depth, a lateral image (optical section) can be formed by translating the beam; the spot size of the focused beam (easily less than 10 xcexcm) determines the lateral resolution.
History of Reflectometry
When optical fibers were introduced into the communications industry in the 1970s, the need immediately arose for a method of testing and locating flaws in fiber cables. The first reflectometers (Barnoski and Jensen, 1976), which operated in the time domain, simply measured the round trip time of flight to a reflecting fiber flaw. Typical pulse widths were a few nanoseconds, so spatial resolution was about one meter.
In the 1980s there appeared low-coherence reflectometers which operate in the frequency domain (Danielson and Whittenburg, 1987; Takada et al., 1987; Youngquist et al., 1987). In this technique a spectrally broad (30 nm) light source operating in the near infrared (800 to 1300 nm) is employed in a Michelson interferometer, one leg of which is the fiber under test. The light source has a coherence time of 70 femtoseconds, a considerable improvement over the timedomain pulse widths. As the reference path length is varied, the interferometer output is monitored for interference fringes that occur when light is reflected or back-scattered from a point a distance along the tested fiber equal to the reference path length. The spatial resolution along the tested fiber is one-half the coherence length because the fiber is traversed twice in that leg of the interferometer. (Actually the geometrical spatial resolution is even smaller by a factor of n, where n is the refractive index of the fiber.) For a spectral width of 30 nm, the geometrical spatial resolution along a fiber is 7 xcexcm.
Shortly thereafter ophthalmologists adapted this low-coherence reflectometer to measure the length of the eye (Fercher et al., 1988; Hitzenberger, 1991). Finally lateral scans were added, and both lateral and depth data were interpreted in terms of images of the sample, usually 2-D images with one lateral and one depth dimension (Huang et al., 1991 a,b). The image presumably represents the spatial variation of the optical properties of the sample, primarily the scattering coefficient.
Polarization Effects
Interference occurs at the output of the OCM only between the same polarization components of the electric fields returning from the reference mirror and the sample, respectively. Birefringence effects in the optical fibers or in the sample may alter the relative magnitude and phase of the two polarization components emitted by the source and hence reduce the amplitude of the interference fringes at the photodetector. To eliminate problems in the fibers, some workers have used polarization-preserving fibers and linearly polarized light to eliminate polarization-dispersion effects that lead to different optical path lengths for different polarization states (Clivaz et al., 1992). Kobayashi et al. (1991) constructed a polarization-insensitive reflectometer by separating the two polarization states at the output of the interferometer and measuring their interference fringes with two independent detectors. The sum of the detector outputs is independent of birefringence effects in the fibers or the sample. On the other hand, Wang et al. (1994) devised a simple, inexpensive means of circumventing birefringence effects. They judiciously twist the reference fiber, introducing stress birefringence, until the polarization states of the reference and sample fields are matched and the amplitude of the interference fringes is maximized. Rather than compensate for and eliminate birefringence effects, Hee et al. (1992) have constructed a low-coherence reflectometer to exploit polarization changes in the sample. With this device they were able to measure the birefringence properties of a calf coronary artery.
The present invention provides a high resolution optical coherence microscope system for visualizing structures below a surface of a biological sample. The system includes a light source emitting light in a wavelength of between 700 and 1500 nm, the light being directed along a sample path and a reference path. The length of at least one of the paths is a modulated path having a selected amplitude of modulation that is equal to or less than about 3 fringes of the wavelength. The modulation may occur at a frequency of at least about 50 kHz, 100 kHz, 300 kHz or at a higher frequency. The light directed along the sample path may scan the biological sample, the scan resulting in an image of a portion of the biological sample; the portion may be between about 100 xcexcm and about 4000 xcexcm below the surface of the sample.
The image may include one or more layers. Each layer may be derived from multiple voxels all corresponding to substantially the same depth below the surface of the sample. The image may include at least about 50 distinct layers, each of the layers derived from a distinct group of voxels, with all voxels for each distinct layer corresponding to substantially the same distinct depth below the surface of the sample. The image likewise may include blended voxels of several layers, such that the image may be a three-dimensional rendering of the portion of the biological sample. The OCM system of the invention further may include a coherence volume about a plane at which the length of the sample is equal to the length of the reference path, such that the coherence volume exists below the surface of the biological sample.
The light from the sample path may enter the sample and taper to a beam waist diameter of not more than 20 xcexcm within the sample. The beam waist is coincident with the coherence volume, such that resolution of structures within the sample is a distance less than or equal to the diameter of the beam waist.
The invention further provides a method of visualizing a structure beneath a surface of a biological sample, employing the OCM system described herein. The OCM system also allows a method of analyzing a biological function based on visualization of in vivo changes in structures beneath a surface of a biological sample. The function to be analyzed may include, for example, gene regulation, development, messenger response, and stress.
The invention also provides a method of visualizing a structure beneath a surface of a biological sample. The method may include the steps of: providing light having a wavelength between 700 and 1500 nm; dividing the light into a sample light path and a reference light path; modulating the length of at least one of the light paths at an amplitude no greater than about 3 fringes of the wavelength; directing light from the sample path into the biological sample, such that the light tapers to a beam waist at a selected depth below the surface of the sample, and such that the beam waist is coincident with a coherence volume about a plane of equal path length of the sample path and the reference path; and detecting an image at the selected depth below the surface of the sample to visualize the structure.
In accordance with this method, the directing step may be repeated at least 100 times, and after each directing step, the method may include the additional step of translocating the sample light path to a different position in the biological sample. The image thus visualized may indicate a difference between a mutant biological sample and a non-mutant biological sample. The image may include a pattern of light scatter, wherein the pattern correlates with a characteristic of the biological sample, such as, for example, gene activity, differentiation, cell elongation, cell dormancy, stress response, and pathogen response.